Reversible post-translational modification of proteins by ubiquitin (Ub) is a regulatory mechanism that controls nearly all aspects of cellular function i eukaryotes. Ub conjugation alters properties of the target protein such as its stability, subcellulr localization, intermolecular interactions, and conformation/activity. It is through a combination o these effects that Ub conjugation to key target proteins regulates processes such as cell cycle control, signal transduction, immunity, and differentiation. The relevance of the Ub pathway to human health is underscored by the fact that its dysregulation is implicated in pathologies such as cancers, neurological disorders, cardiovascular disease, and immune disorders and that it is a validated target for therapeutic intervention in cancer with FDA-approved medications extending the lives of multiple myeloma patients. Ub conjugation to target proteins proceeds through the sequential interactions and activities of three enzymes, E1, E2, and E3. E1 performs two main functions: (1) activation of Ub in a two-step process that results in the formation of a thioester bond between Ub and the E1 catalytic cysteine, and (2) recruitment of E2 enzymes followed by transfer of Ub to an E2 catalytic cysteine (thioester transfer). After E1-E2 thioester transfer, the resulting E2-Ub intermediate interacts with members of three different families of Ub E3 ligases that catalyze Ub conjugation to target proteins by distinct mechanisms. Prior to catalysis of Ub conjugation by RING-between-RING (RBR) E3 family members, Ub must be transferred from the E2 catalytic cysteine to an RBR E3 catalytic cysteine in a process that is mechanistically analogous to E1-E2 thioester transfer, but the structural basis for which i unknown. In humans, there are two Ub E1s (Uba1 and Uba6) that exhibit overlapping but distinct specificities for more than 30 different Ub E2s and there are ~12 RBR E3 family members that function with overlapping but distinct subsets of E2-Ub intermediates. Specificity is essential to the integrity of Ub signaling, however, poor conservation across E1s, E2s, and RBR E3s at predicted contact sites suggests structural plasticity at E1-E2 and E2-RBR interfaces that precludes our ability to establish the rules governing specificity based on sequence analysis. Through use of biochemical, biophysical, and structural approaches, the research in this proposal aims establish the rules governing molecular recognition in E1-E2 (Aim 1) and E2-RBR E3 (Aim 2) interactions and to determine the structural basis by which Ub is transferred from E1 to E2 to RBR E3 prior to finally being conjugated to the target protein.